We previously compared the inhibition, insertion, and exonucleolytic removal of five currently approved antiviral nucleotide analogs on the purified human recombinant DNA polymerase gamma. The apparent Km and kcat values were determined for the incorporation of TTP, dCTP, dGTP, 2-3-dideoxy-TTP (ddTTP), 3-azido-TTP (AZT-TP), 2-3-dideoxy-CTP (ddCTP), 2-3didehydro-TTP (D4T-TP), (-)-2,3-dideoxy-3-thiacytidine (3TC-TP), and carbocyclic 2,3-didehydro-dGTP (CBV-TP). Human pol gamma readily incorporated all five analogs into DNA but with varying efficiencies. Kinetic studies indicate that the apparent in vitro hierarchy of mitochondrial toxicity for the approved NRTIs is: ddC(zalcitabine) 8805; ddI(didanosine) 8805; D4T(stavudine) > >3TC(lamivudine) >PMPA(tenofovir)> AZT(zidovudine) > CBV(abacavir). The human pol gamma utilized dideoxynucleotides and D4T-TP in vitro as efficiently as the natural deoxynucleoside triphosphates, whereas AZT-TP, 3TC-TP and CBV-TP were moderate inhibitors of chain elongation. With the exception of terminally incorporated 3TC, the pol gamma 3-5 exonuclease was inefficient at removing these five analogs from DNA and removal required enzyme levels exceeding substrate concentrations. Even though discrimination against inserting AZT and CBV makes them only moderate inhibitors in vitro, their inefficient excision suggest AZT and CBV may persist in vivo once incorporated into mtDNA by pol gamma. Finally, we found that the exonuclease activity is inhibited by AZT-monophosphate at concentrations known to occur in cells. Thus, although these analogs exert their greatest effect by insertion and chain termination of DNA synthesis, the persistence in DNA and inhibition of proofreading activity may also contribute to mitochondrial toxicity. We are continuing to utilize presteady state kinetic analysis to measure the incorporation and the selectivity of new antiviral nucleoside analogs by the human pol gamma. Also, animal and tissue culture models of mitochondrial dysfunction are being pursued to evaluate the sensitivity to antiviral therapy. Finally, we are developing novel next generation sequencing assays to investigate the consequence of antiviral treatment of human cells on the integrity of the mitochondrial genome.